Apolipophorin III ‐ lipopolysaccharide interaction using tyrosine fluorescence

Abstract

Apolipophorin III (apoLp-III) is a model exchangeable apolipoprotein, and plays a crucial role in lipid transport processes. ApoLp-III also interacts with lipopolysaccharides (LPS), and may function as a pattern recognition protein. While the lipid A portion of LPS is involved in the binding interaction, apoLp-III shows a strong interaction with the LPS carbohydrate portion. To study the LPS binding site in more detail, binding studies were performed using the intrinsic Tyr fluorescence properties of apoLp-III of Galleria mellonella. The unique Tyr-142 residue is highly quenched in the unbound state, but becomes unquenched when bound to LPS. We showed that this unquenched state exists at pH 7-9, while at a pH < 7 Tyr becomes unquenched. Therefore all binding assays were performed in phosphate buffered saline at pH 7.4. The binding of apoLp-III to a truncated version of LPS (Rd), which lacks the O-antigen, was increased 20-fold compared to LPS, indicating that core sugars play a key-role in binding. Hexoses, found in the core of LPS, were incubated with LPS to compete for apoLp-III binding. A 60% decrease in Tyr fluorescence was observed with glucose and N-acetyl-glucosamine. This indicates that the core sugars of LPS are primarily responsible for apoLp-III binding interaction. This work was supported by grants from the National Institutes of Health (R15 HL077135 and S06 GM 063119-05).